Uncovering the profile of mutations of transforming growth factor beta-induced gene in Chinese corneal dystrophy patients

AIM： To uncover the mutations profile of transforming growth factor beta-induced （TGFBI） gene in Chinese corneal dystrophy patients and further investigate the characteristics of genotype-phenotype correlations. METHODS： Forty-two subjects （6 unrelated families including 15 patients and 8 unaffected members, and 19 sporadic patients） of Chinese origin were subjected to phenotypic and genotypic characterization. The corneal phenotypes of patients were documented by slit lamp photography. Mutation screening of the coding regions of TGFBI was performed by direct sequencing. RESULTS： We detected four corneal dystrophy types. The most frequent phenotypes were granular corneal dystrophy （GCD） （including 3 families and 8 sporadic patients） and lattice corneal dystrophy （LCD） （including 2 families and 9 sporadic patients）. The next phenotypes were corneal dystrophy of Bowman layer （CDB） （1 family and 1 sporadic patient） and epithelial basement membrane dystrophy （EBMD） （1 sporadic patient）. Six distinct mutations responsible for TGFBI corneal dystrophies were identified in 30 individuals with corneal dystrophies. Those were, p.R124H mutation in 1 family and 2 sporadic patients with GCD, p.R555W mutation in 2 families and 3 sporadic patients with GCD, p.R124C mutation in 2 families and 7 sporadic patients with LCD, p.A620D mutation in 1 sporadic patient with LCD, p.H626R mutation in 1 sporadic patient with LCD, and p.R555Q in 1 family and 1 sporadic patient with CDB. No mutation was detected in the remaining 3 atypical GCD patients and 1 EBMD patient, CONCLUSION： GCD and LCD are the most frequent phenotypes in Chinese population. R555W was the most common mutation for GCD; R124C was the most common mutation for LCD, Our findings extend the mutational spectrum of TFGBI , and this is the extensively delineated TGFBI mutation profile associated with the various corneal dystrophies in the Chinese population.

Analysis of the Phenotype and the Restriction Enzyme Mapping Level of Mutations Induced by the New Mutagen Glycidyl Methacrylate

Glycidyl methaerylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMAbound pBR322 were used to transform Eschenchia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, Ap~RTc~S and Ap~STc~R, in the transformants and a deductive mutant Ap~STc~S in the nontranstormants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant Ap~RTc~S, four of seven maps were changed. some sites were shifted to other resistant gene regions, for example, sites of Bgll, EcoRl, Ilindlll. Hinclll, etc., and there was a new recognition site for Hindi (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation. (C)1990 Academic Press, Inc.

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

AIM：To report a phenotypic variant pedigree of lattice corneal dystrophy（LCD）associated with two mutations,R124C and A546 D,in the transforming growth factor betainduced gene（TGFBI）.METHODS：A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction（PCR）of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS：Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION：We observed a novel LCD family which carried two pathogenic mutations（R124C and A546D）in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease,some different modifier alleles may influence the phenotype.

Breeding Sorghum Using Induced Mutations: Future Prospect for Namibia

In arid and semi-arid regions of the world sorghum stands out as a climate change-ready crop with high potential for the production of food, feed, fodder, fiber and fuel in the face of increasing human population. The present review highlights induced mutation breeding technique as a potential tool for improving sorghum in Namibia. The review discussed the following issues;crop improvement using mutagens, mutant screening, selection and evaluation, impact of induced mutation breeding, factors for declining production and future implication of sorghum mutation breeding. In Namibia, severe drought stress resulting in total crop failure has become frequent. This is partly a consequence of farmers growing crop varieties which cannot withstand impact of drought. As such Namibia has limited drought tolerant varieties available for the diverse agro-ecologies. Farmers keep growing the familiar landraces which performs well in good rainfall years but fails to produce stable yield with irregular and erratic rainfall. Thus, breeding new sorghum varieties of high yield and quality combined with multiple agronomic traits including pest and disease resistance and high efficiency in nutrient and water use is needed. Induced mutation is one of the breeding methods utilized worldwide to supplement conventional breeding for developing superior varieties with desirable traits in different crops. Development of high yielding, drought tolerant, and dwarf sorghums with early maturity enables effective utilization of available soils moisture and in optimizing plant density for achieving higher yield in farmers’ fields. Recombination breeding through exploitation of natural genetic variability and mutation breeding to reduce the plant height without disturbing agronomic superiority of elite lines is recommended for sorghum improvement in Namibia.

International Symposium on “Induced Mutations in Plants” in 2008

The year 2008 will mark the 80 th anniversary of mutation induction in plants. The application of mutation techniques has generated a vast amount of genetic variability and has led to the off icial release of at least more than

Induced Mutation for Developing Mutant Rice Lines Tolerant to the Parasitic Weed Striga asiatica (L.) Kuntze

This work aims to screen mutant rice lines tolerant to Striga asiatica.Two rainfed sensitive rice varieties B22 and F154 were used.Plants survival rates of the two parents were significantly lower respectively(9.74a and 11.83a)than those of mutant lines(55.36c to 74.36b);Striga plants emergence/pot were significantly higher for the parents(13.96c and14.89c)compared to the mutants(0.12a to 1.5b);the infection rate of parents(7.37b;7.86b)was higher compared to the mutants(2.27a to 2.74a);fertility rate/plant of parents was lower(20.98%b;22.29%b)but much higher than mutants(72.19%b to 78.35%b);the average panicle number/plant of parents was significantly lower(0.5a;1a)than those of mutants(1.5b to 2.4bc)and the 100 g grain weight of parents are lower(2.35a;2.56a)than those of mutants(3.19b to 3.23b).The culture of those mutant lines may increase rice production and contribute to enhancing food security in Madagascar.

Space-flight Mutation of Streptomyces gilvosporeus for Enhancing Natamycin Production

Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the survival ratio was 43.1% and 3.0%, respectively. An improved mutant producing natamycin, S. gilvosporeus LK-45, was screened, which showed natamycin productivity of 1420mg·L^-1. A mutant resistant to 2-deoxy glucose, S.gilvosporeus LK-119, was further obtained using a＇rational screening procedure. The natamycin productivity of 1940mg·L^-1 was achieved when glucose was used as the carbon source.

Mutation Techniques for Gene Discovery and Crop Improvement

One of the most important breakthroughs in the history of genetics was the discovery that mutations can be artif icially induced in organisms (van Harten, 1998). Artif icially induced mutations, by physical and chemical muta-

Genomic integrity of human induced pluripotent stem cells:Reprogramming, differentiation and applications

Ten years after the initial generation of induced pluripotent stem cells(hiPSCs)from human tissues,their potential is no longer questioned,with over 15000 publications listed on PubMed,covering various fields of research;including disease modeling,cell therapy strategies,pharmacology/toxicology screening and 3D organoid systems.However,despite evidences that the presence of mutations in hiPSCs should be a concern,publications addressing genomic integrity of these cells represent less than 1%of the literature.After a first overview of the mutation types currently reported in hiPSCs,including karyotype abnormalities,copy number variations,single point mutation as well as uniparental disomy,this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity.Then,a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest.Finally,in a last section,the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed.We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.

Induced Resistance to <i>Striga hermonthica</i>in Sorghum by Gamma Irradiation

Striga species affect the potential productivity of cereals in sub-Saharian Africa due to the lack of durable Striga-resistance in host crops. This study aimed at inducing the new source of resistance in sorghum using gamma irradiation. Dry seeds of three Sorghum varieties;Grinkan, ICV1049 and Sariaso14 were gamma-irradiated with 200 Gy, 300 Gy, 400 Gy and 500 Gy. Screening strategies involved a 2-year field and greenhouse experiments, where mutant Sorghum families, their parents and resistant control were artificially infected with Striga hermonthica seeds. Field screenings revealed induced genetic variability among them, forty families significantly reduced the number of emerged Striga plants or showed good Sorghum grain yield performance despite the infection by S. hermonthica ecotype from Burkina Faso. The induced putative resistant mutants were identified across the four applied gamma-irradiation doses. Greenhouse experiment confirmed Striga resistance in seven mutant Sorghum families leading to no emergence of Burkina’s S. hermonthica ecotype along with high resistance index (RI) and low Striga damage score. Among them, two mutants SA38M5 and IC47M5 withstood S. hermonthica ecotype from Sudan. The induced mutants will be evaluated for the release to farmers for commercial production. Further studies are ongoing on confirmed mutants to highlight their Striga resistance mechanisms and explore the potential of pyramiding different mechanisms to produce durable resistance to S. hermonthica in sorghum.

Patient-specific induced pluripotent stem cells as“disease-in-adish”models for inherited cardiomyopathies and channelopathies–15 years of research

Among inherited cardiac conditions,a special place is kept by cardiomyopathies(CMPs)and channelopathies(CNPs),which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality.Like other inherited cardiac conditions,genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant,challenging our understanding of the underlying pathogenic mechanisms.Until recently,the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms.The advent of induced pluripotent stem cell(iPSC)technology,along with advances in gene editing,offered unprecedented opportunities to explore hereditary CMPs and CNPs.Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers,genetic identity with the subject from whom they were derived,and ease of gene editing,all of which were used to generate“disease-in-a-dish”models of monogenic cardiac conditions.Functionally,iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype,allowed the study of disease mechanisms in an individual-/allele-specific manner,as well as the customization of therapeutic regimen.This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms,personalized therapy and deoxyribonucleic acid variant functional annotation.

Presenilin mutations and their impact on neuronal differentiation in Alzheimer’s disease

The presenilin genes(PSEN1 and PSEN2)are mainly responsible for causing early-onset familial Alzheimer’s disease,harboring~300 causative mutations,and representing~90%of all mutations associated with a very aggressive disease form.Presenilin 1 is the catalytic core of theγ-secretase complex that conducts the intramembranous proteolytic excision of multiple transmembrane proteins like the amyloid precursor protein,Notch-1,N-and E-cadherin,LRP,Syndecan,Delta,Jagged,CD44,ErbB4,and Nectin1a.Presenilin 1 plays an essential role in neural progenitor maintenance,neurogenesis,neurite outgrowth,synaptic function,neuronal function,myelination,and plasticity.Therefore,an imbalance caused by mutations in presenilin 1/γ-secretase might cause aberrant signaling,synaptic dysfunction,memory impairment,and increased Aβ42/Aβ40 ratio,contributing to neurodegeneration during the initial stages of Alzheimer’s disease pathogenesis.This review focuses on the neuronal differentiation dysregulation mediated by PSEN1 mutations in Alzheimer’s disease.Furthermore,we emphasize the importance of Alzheimer’s disease-induced pluripotent stem cells models in analyzing PSEN1 mutations implication over the early stages of the Alzheimer’s disease pathogenesis throughout neuronal differentiation impairment.

盾尾密封失效诱发砂土地基盾构管片环失稳坍塌研究

盾构管片环失稳坍塌案例时有发生,探究其失稳坍塌发生条件对于预防重大安全事故发生具有重要意义。基于Midas GTS建立30环考虑环缝螺栓作用的荷载−结构计算模型,从盾尾管片环地基掏空和盾尾姿态突变2个方面探讨盾构管片环结构失稳破坏过程及失稳坍塌的发生条件。结果表明:盾尾渗漏使盾尾壳体失去了周围地基的有效约束作用,盾尾发生前仰后俯的姿态变化,隧道结构发生横向“横鸭蛋”和纵向挠曲变形,最终导致部分管片环坍塌。盾尾下沉位移是诱发管片环失稳坍塌的主要因素,盾尾下沉导致隧道纵向变形快速发展,快于盾尾渗漏引起管片环周围地基掏空所导致的隧道纵向变形。当盾尾下沉位移大于0.50 m且掏空范围大于5环、盾尾下沉位移大于0.45 m且掏空范围大于10环、盾尾下沉位移大于0.40 m且掏空范围大于11环时,环缝最大张开量超过单根螺栓极限应力时的环缝张开量47.43 mm,部分纵向螺栓被拉断,管片外水土发生喷射,加快管片环外砂土快速流失。当盾尾下沉位移大于0.5 m且掏空范围大于9环时,环缝最大张开量超过65.2 mm,管片环椭圆率超过46.1‰,最终诱发多环管片环失稳坍塌。研究结果可为盾尾渗漏诱发的重大安全风险评判提供参考。

狗牙根诱变后代生长速度评价及DNA指纹图谱构建

狗牙根(Cynodon dactylon)是一种常见的暖季型草,利用诱变技术处理现有品种进行改良,是快速选育优良新品种的一种途径。本研究以国审品种‘阳江’狗牙根通过60Co-γ辐射处理获得的12个诱变后代为材料,进行生长速度评价和基于序列相关的扩增多态性(SRAP)分子标记技术的DNA指纹图谱构建。结果表明,诱变后代间匍匐茎总长度、地上部分干重、匍匐茎数量、地下部分干重等4个指标均存在显著的变异。对这4个指标进行隶属函数分析,对诱变后代的生长速度进行综合评价,认为其生长速度由快到慢依次为M37>M16>M1>M25>M10>M18>M28>M29>M26>‘阳江’>M22>M31>M4。利用12对SRAP引物对12个诱变后代和6个狗牙根主栽品种进行指纹图谱构建,其中7对引物组合可以直接对18份参试材料进行准确鉴定。本研究为进一步国产狗牙根新品种的选育和保护提供了可靠的实验依据。

基于Mann-Kendall的广东省长效避孕服务利用和人工流产的趋势与突变分析

目的了解广东省长效避孕服务利用和人工流产变化趋势,为制定有效的生殖保健服务策略,提高妇女生殖健康提供依据。方法采用Mann-Kendall检验法对2008—2021年广东省长效避孕服务利用总例数、各种长效避孕方法例数与占比、人工流产例数、人工流产活产比进行趋势和突变分析。结果2008—2021年广东省长效避孕服务利用总例数呈现下降趋势,长效不可逆避孕方法例数呈先增后降的趋势、长效可逆避孕方法例数呈现下降趋势;这三个指标突变点分别出现在2016、2018、2014年。各种长效避孕方法服务利用上,放置宫内节育器术占67.3%,输卵管绝育术占29.1%,输精管绝育术占3.5%,放置皮下填埋剂术占0.1%。全省人工流产例数呈下降趋势,突变点出现在2018年;人工流产活产比相对稳定,年均人工流产活产比为76.5%。从区域层面看,全省与珠三角地区长效可逆避孕方法例数和人工流产例数的变化趋势呈现出平行关系,粤东、粤西和粤北地区长效可逆避孕方法例数变化趋势与人工流产例数变化趋势的关联各异。结论广东省放置宫内节育器术在长效避孕方法中占主导地位,输卵管绝育术次之,长效避孕责任主要由女性群体承担。区域间长效可逆避孕方法与人工流产变化趋势呈现出不同的关系,有必要深入了解导致这些地区差异的因素,促进不同区域避孕服务的有效落实,避免发生意外妊娠。此外,建议地区积极探讨将避孕服务与妇女全生命周期服务整合,提高妇女生殖健康水平。

Pool-ARMS:一种高效检测混合遗传样本中特定单核苷酸变异的方法

单核苷酸变异(SNV)是控制人类疾病和动植物经济性状的遗传基础之一,经济、简便和高效的检测体系可助力遗传病筛查和植物碱基编辑与诱变群体中携带SNV的个体的定向筛选。本研究根据扩增阻滞突变系统PCR(ARMS-PCR)的原理,设计了一种适合在水稻混合样本中筛选特定SNV个体的方法 Pool-ARMS。该方法的要点是:设计聚合酶链式反应(PCR)引物、建立特异性扩增携带SNV片段的PCR程序;分析不同组织和样品混合比例提取DNA的扩增效果;建立筛选特定SNV的Pool-ARMS体系。以控制光周期敏感性雄性不育(PGMS)水稻的两个基因为例,通过优化PCR引物和反应程序,建立了利用叶片和芽鞘组织混样提取DNA检测突变的体系,前者突变检出水平为1∶49,后者为1∶24。利用该技术体系对63份碱基编辑水稻幼苗进行检测,结果与Sanger测序分析一致。本研究建立的Pool-ARMS方法有望应用于包括水稻在内的动植物单碱基编辑群体和理化诱变群体中目标变异的定向筛选。

黄瓜基于扩增子测序的TILLING技术体系的建立

定向诱导基因组局部突变(TILLING)技术能够对突变群体中的点突变进行快速筛选,而样品混池深度是影响其效率的主要因素。为提高TILLING的突变检测效率,本研究以甲基磺酸乙酯(EMS)诱变群体构建的突变体库为基础,对其中部分M2代植株进行突变性状观察,结果表明表型突变率为10.80%。同时利用已知矮化突变体与野生型进行混池,分别构建96、192、288、384四个不同深度的测序池,使用GATK软件进行突变检测,为对假阳性位点进行有效过滤,候选突变位点分别根据突变类型及归一化深度作为阈值两次进行过滤。结果表明,96~288倍的混池深度均能稳定检出预设的已知突变位点,并与Sanger测序结果相一致。与前人相比,本研究通过优化混池深度与过滤阈值,实现了更高混池深度的突变检测,并在黄瓜中建立了基于扩增子测序的TILLING技术体系,可实现更高通量地对大规模突变群体进行目标突变的鉴定。

Pharmacologic inducers of the uric acid exporter ABCG2 as potential drugs for treatment of gouty arthritis

Uric acid is the end product of purine catabolism and its plasma levels are maintained below its maximum solubility in water(6–7 mg/dl).The plasma levels are tightly regulated as the balance between the rate of production and the rate of excretion,the latter occurring in urine(kidney),bile(liver)and feces(intestinal tract).Reabsorption in kidney is also an important component of this process.Both excretion and reabsorption are mediated by specific transporters.Disruption of the balance between production and excretion leads to hyperuricemia,which increases the risk of uric acid crystallization as monosodium urate with subsequent deposition of the crystals in joints causing gouty arthritis.Loss-of-function mutations in the transporters that mediate uric acid excretion are associated with gout.The ATP-Binding Cassette exporter ABCG2 is important in uric acid excretion at all three sites:kidney(urine),liver(bile),and intestine(feces).Mutations in this transporter cause gout and these mutations occur at significant prevalence in general population.However,mutations that are most prevalent result only in partial loss of transport function.Therefore,if the expression of these partially defective transporters could be induced,the increased number of the transporter molecules would compensate for the mutation-associated decrease in transport function and hence increase uric acid excretion.As such,pharmacologic agents with ability to induce the expression of ABCG2 represent potentially a novel class of drugs for treatment of gouty arthritis.

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Targeting Induced Local Lesions IN Genomes （TILLING） is a reverse genetics strategy for the high-throughput screening of induced mutations.γ, radiation, which often induces both insertion/deletion （Indel） and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting （HRM） analysis. Pooled rice （Oryza sativa） samples mixed at a 1：7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ, ray-induced mutants in rice. For demonstration, a γ, ray-mutagenized M2 rice population （n=4560） was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution （G→A） and one single nucleotide insertion （A） were identified in OsLCT1, and one tdnucleotide （TTC） deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ, ray mutagenesis to the breeding of rice and other seed crops.

Induced pluripotent stem cell-derived motor neurons from amyotrophic lateral sclerosis(ALS)patients carrying different superoxide dismutase 1 mutations recapitulate pathological features of ALS

Background:Investigations of the pathogenic mechanisms in motor neurons(MNs)derived from amyotrophic lateral sclerosis(ALS)disease-specific induced pluripotent stem(iPS)cell lines could improve understanding of the issues affecting MNs.Therefore,in this study we explored mutant superoxide dismutase 1(SOD1)protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations.Methods:We generated induced pluripotent stem cell(iPSC)lines from two familial ALS(FALS)patients withSOD1-V14M andSOD1-C111Y mutations,and then differentiated them into MNs.We investigated levels of the SOD1 protein in iPSCs and MNs,the intracellular Ca2+levels in MNs,and the lactate dehydrogenase(LDH)activity in the process of differentiation into the MNs derived from the controls and ALS patients’iPSCs.Results:The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers.We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the twoSOD1 mutant patients.However,at no time did we observe stronger LDH activity in the patient lines compared with the control lines.Conclusions:MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis,providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy.Incremental mutant expressions of SOD1 in MNs may have disrupted MN function,either causing or contributing to the intracellular calcium disturbances,which could lead to the occurrence and development of the disease.